1. The inositol phosphatase SHIP-1 is negatively regulated by Fli-1 and its loss accelerates leukemogenesis
by Gurpreet K. Lakhanpal, Laura M. Vecchiarelli-Federico, You-Jun Li, Jiu-Wei Cui, Monica L. Bailey, David E. Spaner, Daniel J. Dumont, Dwayne L. Barber, and Yaacov Ben-David
Blood
Volume 116(3):
July 22, 2010
©2010 by American Society of Hematology

2. SHIP-1 Fli-1 protein expression are inversely correlated in mouse erythroleukemia cells.
SHIP-1 Fli-1 protein expression are inversely correlated in mouse erythroleukemia cells. SHIP-1, Fli-1 (A) and PTEN (B) expression levels in HB1.1, CB3, HB9.2, and HB22.2 cells, derived from spleens of F-MuLV–infected Balb/c mice, and HB60-5, DP16-1, and DP17-17 cells, derived from spleens of FV-P (SFFV-P + F-MuLV)-infected DBA/2J adult mice. SHIP-1 and Fli-1 expression levels in (C) the spleens of 8-week-old F-MuLV–infected mice (B2-B4). (D) Exogenous expression of Fli-1 in DP17-17 cells (MigR1 Fli-1) results in decreased expression of SHIP-1 compared with nontransduced (N/T) and empty vector control (MigR1) infected cells. β-Actin was used as a loading control for all samples.
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology

3. Acceleration of F-MuLV–induced erythroleukemia progression in SHIP-1 deficient mice.
Acceleration of F-MuLV–induced erythroleukemia progression in SHIP-1 deficient mice. (A) Mean survival rate of F-MuLV–infected Balb/c mice. SHIP-1−/− n = 11; SHIP-1+/− n = 25; SHIP-1+/+ n = 21. (B) The survival rate of mice infected with F-MuLV as presented by a Kaplan-Meier survival curve. (C) Fli-1 and SHIP-1 protein expression in the spleens of 8-week-old SHIP-1−/− and SHIP-1+/− F-MuLV-induced erythroleukemic mice. (D) Mean hematocrit levels of 8-week-old mice infected with F-MuLV. (E) Mean RBC counts of 8-week-old mice infected with F-MuLV. (F) Mean hemoglobin levels in 8-week-old mice infected with F-MuLV. (D-F) n = 3.
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology

4. Down-regulation of SHIP-1 in HB60-5 cells blocks differentiation and accelerates proliferation.
Down-regulation of SHIP-1 in HB60-5 cells blocks differentiation and accelerates proliferation. (A) RNAi-mediated down-regulation of SHIP-1 in HB60-5 double-sorted population (dbl sort) and clone 8D cell populations results in increased Fli-1 expression and increased phosphorylation of AKT and MAPK compared with nonspecific negative control shRNA expressing cells (control). (B) Fli-1 protein expression is reduced in the presence of the PI 3-K inhibitor, LY294002, but not the MAPK inhibitor, PD (C) Northern blot analysis of RNAi-induced SHIP-1 knockdown cells displays reduced Epo-induced differentiation as determined through hybridization with an α-globin probe. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Double-sorted SHIP-1 shRNA expressing HB60-5 cells (SHIP-1 shRNA-11), grown in the presence of Epo, display accelerated proliferation, compared with negative control shRNA-expressing cells. Cells were maintained in 1 U/mL Epo. This experiment was performed in triplicate.
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology

5. In vitro binding of Fli-1 to the SHIP-1 promoter.
In vitro binding of Fli-1 to the SHIP-1 promoter. (A) The 988-bp promoter region before the start codon of murine SHIP-1 was cloned into the pGL3-enhancer vector. Three possible Ets binding sites (EBS) are highlighted. S1 and S2 indicate the locations of the primers used to amplify the 294-bp region of the SHIP-1 promoter. (B) The SHIP-1 promoter regions of human, mouse, and rat were compared for conservation of nucleotides using BLAST (National Center for Biotechnology Information). The underlined regions (EBS1 and EBS2) are fully conserved in all 3 species. (C) Binding of Fli-1 to the promoters of SHIP-1 (lane 4) and MDM2 (lane 1) as determined by ChIP in CB3, HB60-5, and KH16 erythroleukemic cells using either 2 μg Fli-1 or control rabbit IgG antibody. The MDM2 promoter was used as a positive control, because it is a known Fli-1 target gene.27 (D) ChIP quantitative-PCR in CB3 cells using Fli-1 and normal rabbit IgG antibodies illustrating Fli-1 chromatin occupancy of the indicated gene promoters, as well as a region 3 kb upstream the SHIP-1 promoter (UP-SHIP-1). The β-actin and upstream SHIP-1 promoters were used as negative controls. Results are based on the relative proportions of the input and chromatin precipitated by the Fli-1 and control IgG antibodies, where the input is equal to 1.
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology

6. Fli-1 binds specifically to EBS1 in the SHIP-1 promoter and negatively regulates its expression.
Fli-1 binds specifically to EBS1 in the SHIP-1 promoter and negatively regulates its expression. (A) Nuclear extracts from CB3 cells were incubated with EBS1-3 sites in the presence or absence of the Fli-1 antibody and nonspecific competitor poly (dI-dC) and subjected to EMSA. Competition assays were performed in the presence of 10-fold excess unlabeled oligonucleotides (cold competitor). (B) Two nucleotides in EBS1 were changed from ACAGGAAGTCA to ACAGGTTGTCA (designated MUT-EBS-1). Nuclear extracts from CB3 cells were incubated in the presence of poly (dI-dC) and γ-32P-labeled oligonucleotides containing EBS1, MUT-EBS1, or MDM227 and subjected to EMSA and supershifting with the Fli-1 antibody. Competition assays were performed in the presence of 100-fold excess unlabeled oligonucleotides. (C) Luciferase assays were performed in 293T cells cotransfected with the indicated amounts of a Fli-1-expression vector, either the pGL3-SHIP-1 or pGL3-mut-SHIP-1 vector and the Renilla luciferase vector. The pGL3-SHIP-1 luciferase reporter vector contains a 988-bp region within the SHIP-1 promoter (Figure 4A). Site-directed mutagenesis was used to alter nucleotides ACAGGAAGTCA to ACAGGTTGTCA in EBS1 of pGL3-mut-SHIP-1. The relative luciferase units (RLU) are representative of the firefly luciferase/Renilla luciferase signals (×100). Luciferase assays were performed in triplicates. (D) Fli-1 protein expression in 293T cells transfected with the indicated amounts of the Fli-1 expression vector, relative to HB60-5 and CB3 cells. The 2 Fli-1 protein products are observed as a result of 2 isoforms, 48 and 51 kDa, synthesized by alternative translation initiation through the use of 2 highly conserved in-frame initiation codons.47
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology

7. Negative feedback loop model of Fli-1 and SHIP-1 regulation.
Negative feedback loop model of Fli-1 and SHIP-1 regulation. During Epo-induced differentiation of erythroid progenitor cells, SHIP-1 expression is high and is able to negatively regulate the PI 3-K/Akt pathway, leading to lower Fli-1 expression. Upon activation of Fli-1 as a result of insertional mutagenesis, the PI 3-K/Akt and the Ras/MAPK pathways are activated. Increased activity of Akt through the PI 3-K/Akt and/or Ras/PI 3-K/Akt pathways further increases transcription of fli-1, leading to a block in differentiation and acceleration of proliferation by Epo. As Fli-1 levels increase, SHIP-1 transcription is suppressed to a greater extent, eventually resulting in complete repression in late stage erythroleukemia.
Gurpreet K. Lakhanpal et al. Blood 2010;116:
©2010 by American Society of Hematology